Neural tube defects (NTDs) are common, costly, and deadly human congenital anomalies whose etiologies remain largely unknown. One of the most promising clues to the causes of NTDs is that women who use vitamins containing folic acid in early pregnancy are at much lower risk for NTD-affected pregnancies. However, the underlying mechanisms by which folic acid contributes to these reduced risks are unknown. The most commonly held hypothesis is that folate intake prevents NTDs by compensating for individual genetic susceptibilities, although clear identification of genetic determinants through association studies has proven elusive. The approach we propose to further define the molecular genetic mechanisms behind neural tube defects and folate-prevention therapy is rooted in the results of a pilot study to identify nonsynonymous substitutions in vitamin-dependent enzymes that impair function, yet are augmentable by elevated vitamin concentration. This pilot has demonstrated that deep sequencing reveals a substantial amount of low frequency, nonsynonymous variation in folate pathway genes (frequencies <=1%) that has gone unnoticed thus far. Furthermore, using a robust assay platform based on complementation in S. cerevisiae, we have demonstrated that approximately one-half of these low frequency variants affect enzyme function and have identified novel folate-remedial alleles. We hypothesize that genetic susceptibilities in folate metabolism can be etiological for NTDs and that these susceptibilities can be conferred by both low-frequency and common variants and by the possible synergy between these. To test this hypothesis, we will resequence the coding regions in 19 folate metabolic genes from a population of 250 NTD-affected infants and 250 controls, for which we also have information on maternal nutritional intake. We will test all enzyme variants for functional impact and folate remediation, and correlate functional studies with clinical phenotype and nutritional data. We hope to better define the causality of NTDs, understand the remedial role of folate supplementation, and determine whether additional folate supplementation may be preventative. We have assembled a unique combination of scientific expertise to execute this research plan. Relevance: This proposal should reveal the causes of a common form of birth defect known as neural tube defects. Ultimately, this research may lead to better diagnostic and preventive strategies.